There is a pressing national need for an improved vaccine against Bacillus anthracis, the causative agent of anthrax, that can be used for prophylactic mass inoculation as well as immediately after release of a biological weapon. An ideal vaccine will be easily formulated at high consistency and purity, will not require a cold chain for storage and transport, and will be deliverable by a needle-free method. It will have strong adjuvant properties and be based on a platform accommodating the inclusion of multiple subunits. Finally, this vaccine will protect against the earliest stages of the disease (e.g., by recognizing the spore in the lung mucosa, prior to uptake by phagocytic cells). To do this, the vaccine must target epitopes on the spore surface. If the same vaccine also targeted protective antigen (PA), the major component of the existing vaccine, then initiation of anthrax, as well as its later stages, might be prevented. There is already evidence that this strategy is very likely to result in a superior systemic response, since spore components have been shown to enhance a PA-based vaccine. In this proposal, we will generate a novel B. anthracis vaccine, directed against multiple spore-surface antigens and protective antigen (PA), using nanolipoprotein particle (NLP) technology. NLPs are self-assembled, nanometer-sized disk-shaped particles made from purified apolipoprotein and lipid reagents. Because they can be readily engineered to incorporate and display almost any protein, NLPs are an ideal platform for presenting antigens to the immune system as a vaccine. We will leverage prior research in our laboratories, identifying spore-surface proteins and creating nickel-chelated nanolipoprotein particles, to generate nanoparticles bearing PA as well as at least three spore proteins and test their ability to function as a nasal vaccine protecting mice from a challenge with virulent spores. We have the following specific aims: 1) Generate and analyze a nickel-chelated nanolipoprotein particle (NiNLP) vaccine bearing PA and the three known spore-surface proteins, BclA, BclB, ExsK. We will formulate this vaccine, analyze the mucosal and systemic immune responses that it stimulates, and measure its ability to protect against a challenge with virulent Ames strain B. anthracis spores. 2) Generate and test an NiNLP vaccine against spores manipulated to defeat anti-spore vaccines. The outermost spore structure, called the exosporium, could be easily removed by an enemy, thereby defeating an anti-spore vaccine. Therefore, we will generate a vaccine that includes proteins on the surface (the coat) of spores lacking the exosporium. We will incorporate a known coat-surface protein and identify additional coat-surface proteins for inclusion into the vaccine. We will analyze them immunologically and measure their protective efficacy as in Aim 1. Public Health Relevance: Bacillus anthracis spores, the cause of anthrax, are a highly effective biological weapon. We propose to generate a vaccine to protect against anthrax that will be suitable for mass immunization and will be effective against spores engineered to lack the usual surface proteins. This vaccine could not only greatly reduce loss of life in the event of an attack, but could reduce the appeal of the use of the biological weapon.